DESCRIPTION (Adapted from the applicant s abstract): The precise structure of DNA and its topological organization in chromatin has important functional consequences for the regulation of gene expression. Advances in our understanding of histone modifications and non-histone chromosomal proteins that modify and/or interact with histones, the basic protein components of chromatin, are likely to impinge on a variety of basic issues in molecular and cell biology. One long-range goal for this proposal is to carry out an in-depth analysis of linker histone (H1) and H3 phosphorylation. It is proposed that phosphorylation of H1 and/or H3 leads to decondensation of the chromatin fiber, an event causally linked to gene-specific activation. An immediate aim is to understand the consequences of mutations in known in vivo phosphorylation sites in H I and H3 in Tetrahymena and yeast. The investigator will also attempt to identify and characterize the kinase(s) responsible for establishing these modifications, and to identify proteins that potentially interact with these histones in their modified form. In-gel assays have been developed that identify polypeptides displaying kinase activity, or that interact with defined histones or histone amino-terminal "tail" peptides following SDS gel electrophoresis. Work is in progress to obtain microsequence and/or antibodies against these polypeptides. Information from these studies will be used to clone the genes encoding these polypeptides and delete them from the genome to test their in vivo function. A combined biochemical, immunocytological and molecular genetic approach will be used to characterize the role of two heterochromatin-associated polypeptides in gene regulation and programmed DNA elimination. Already, cloning and sequencing two members of this group shows that they are members of the chromodomain family, suggesting unexpected molecular links between heterochromatin assembly and programmed DNA degradation. The unifying goal is to understand how chromatin is modulated to promote gene expression and other cell cycle and developmentally-regulated processes. Understanding these mechanisms in normal cells is essential if the investigator is to understand abnormal development and tumorigenesis.